Screening assays for compounds that cause apoptosis

ABSTRACT

This invention relates to methods of screening for compounds capable of inducing apoptosis in certain tumor cells. The invention also relates to compounds identified by such methods. In addition, the invention relates to methods for the in vitro diagnosis of  Xeroderma pigmentosum  and compounds useful in these methods.

BACKGROUND OF THE INVENTION

[0001] This invention relates to methods of screening for compoundscapable of inducing apoptosis in certain tumor cells. The invention alsorelates to compounds identified by such methods. In addition, theinvention relates to methods for the in vitro diagnosis of Xerodermapigmentosum and compounds useful in these methods.

[0002] Certain tumors, benign, premalignant, and malignant, are known tohave genetic components etiologically. The gene for the nuclearphosphoprotein, p53, is the most commonly mutated gene identified inhuman cancers. Missense mutations occur in tumors of the colon, lung,breast, ovary, bladder, and several other organs. When mutant forms ofthe p53 gene are introduced into primary fibroblasts, these cells areimmortalized. The wild type p53 gene can suppress the growth oftransformed human cells, but oncogenic forms lose this suppressorfunction. Thus, the p53 gene has been termed a “tumor suppressor” gene.

[0003] If the p53 gene of a tumor cell is of the wild type, its p53 geneproduct may nevertheless be interfered with functionally. For example, atransforming viral infection of the cell can interfere with the p53protein product. For instance, certain strains of human papillomavirus(HPV) are transforming and are known to interfere with the p53 proteinfunction because the virus produces a protein, E6, which promotesdegradation of the p53 protein.

[0004] There is also pharmaceutical interest in p53 because p53 proteinis capable of inducing certain tumor cells to undergo apoptosis. Inapoptosis, or “programmed cell death”, a series of lethal events for thecell appear to be generated directly as a result of transcription ofcellular DNA. Thus, apoptosis is a physiologic means for cell death. Forexample, lymphocytes exposed to glucocorticoids die by apoptosis.Involution of hormone sensitive tissue such as breast and prostate thatoccurs when the trophic hormone is removed occurs via apoptosis.

[0005] In particular, recent studies have indicated that theintroduction of wild type (non-mutated) p53 into transformed cell linesthat carry a mutant form of p53 induces the cells to undergo apoptosiswith disintegration of nuclear DNA. It is believed that p53 may suppresstumor development by inducing apoptosis, thus modulating cell growth.

[0006] In addition to p53, there are numerous other genes involved withcell growth. One group of such genes is designated XP because theirderangement can result in the disease Xeroderma pigmentosum. Xerodermapigmentosum is a rare disorder characterized by disfigurement, derangedpigmentation of the skin, scarring and heightened incidence of skincancers, especially on exposure to sunlight. The disease is inherited asan autosomal recessive trait. Eight genetic forms of the disease areknown. Phenotypically these forms vary in their symptoms, signs andseverity. Two of the more grave forms are associated with mentaldeficiencies. These two forms are characterized by mutations in the XPBand XPD genes.

[0007] Selection of drugs for potential therapeutic use against tumorsis an area of medical research which remains fraught with complicationsand which often present an array of suboptimal treatment choices. Thereare currently a multitude of potential compounds available to evaluate.Screening procedures are valuable to limit the bewildering array of drugchoices for further testing. Improvements in screening methods orreagents are highly desirable. In addition, there is a need for betterdiagnosis of XP subtypes. These and other needs are addressed by thepresent invention.

SUMMARY OF THE INVENTION

[0008] The invention provides a method for screening a compound for anability to induce apoptosis. The method includes providing a first cellcontaining either a normal or mutant p53 gene. The first cell isresponsive to p53. For instance, the first cell is typically capable ofundergoing apoptosis after microinjection of a DNA construct expressingwild type p53. The method further includes providing a second cellcontaining at least one mutant Xeroderma pigmentosum gene such as amutant XPB gene, or a mutant XPD gene, or both. The second cell is notusually capable of undergoing apoptosis after microinjection of a DNAconstruct expressing wild type 53. According to a method of theinvention, both the first and second cells are contacted with a compoundof interest. An example of a compound of interest is any compound onedesires to screen for possible use as a chemotherapeutic agent or drug.The method includes detecting whether or not apoptosis of either thefirst or second cell, or both, occurs after contact of the compound tothe cells. A comparison of the observations for apoptosis is made,thereby determining whether the compound can induce apoptosis.

[0009] The first and the second cell can be selected from any of anumber of cell types including benign, premalignant, and malignant. Thefirst and the second cell can be uninfected or infected. If the latter,the infection can be viral, such as from a papilloma virus. The firstand second cell can be selected from any histological or anatomicalclassification. Typically, the cells are selected from the groupconsisting of fibroblastic, epithelial, and hematopoietic cells. Thecells can be derived from a variety of tissues, including tissue ofcolon, lung, breast, ovary, cervix, liver, kidney, nervous system, andhematopoietic system. Preferably, the cells are fibroblastic orlymphoblastic cells.

[0010] The invention also provides a method of screening for a compoundcapable of inhibiting the binding of p53 protein to a Xerodermapigmentosum protein, such as either XPB or XPD proteins or both. Thismethod includes providing a reagent having at least one Xerodermapigmentosum protein, preferably XPB or XPD, or both. The reagent iscontacted with the compound, permitting the compound to compete withwild type p53 protein for a binding site on any or all of the Xerodermapigmentosum protein(s). Subsequently, any binding of the compound to theprotein(s) is detected.

[0011] Additionally, this method can include contacting the reagent withwild type p53 protein and detecting a binding of the wild type p53 to atleast one of the Xeroderma pigmentosum proteins such as an XPB and\orXPD protein(s). The method can further comprise attaching a label to atleast one of the Xeroderma pigmentosum protein(s) and the p53 protein.The label can be any of a number of detectable labels known in the art.Some examples are an antibody, a radioisotope, and a fluorescentmolecule. Conveniently, the reagent has a TFIIH complex containing bothXPB and XPD proteins.

[0012] The invention also provides a method of screening for a compoundcapable of inhibiting at least one Xeroderma pigmentosum helicaseactivity, such as XPB and\or XPD helicase activity. This method includesproviding a reagent having at least one Xeroderma pigmentosum protein,contacting the reagent with the compound which permits the compound tobind to the Xeroderma pigmentosum helicase, and determining the helicaseactivity. Typically, the reagent has a TFIIH complex containing both XPBand XPD proteins.

[0013] The invention also provides compositions. In one embodiment, thecomposition is a compound consisting essentially of the amino acidsequence depicted in Seq. ID No. 2 wherein the compound possesses atleast one, usually two, and preferably all three of the followingproperties: (1) it binds to a binding site on at least one Xerodermapigmentosum helicase, preferably either the XPB helicase or the XPDhelicase or both, (2) it competes with wild type p53 proteins for thebinding site, and (3) it inhibits the helicase activity. Conveniently,the compound is a peptide consisting of the sequence depicted in Seq. IDNo. 2.

[0014] A compound of the invention can be used in a diagnostic method,such as a method of diagnosing a Xeroderma pigmentosum complementationgroup, preferably group B or D, in an individual. Such a method includesproviding a sample cell derived from the individual, contacting thesample cell with a compound of the invention, and detecting whether ornot apoptosis of the sample cell occurs, thereby diagnosing whether ornot the sample cell contains at least one mutant Xeroderma pigmentosumgene, preferably a mutant XPB gene, a mutant XPD gene, or both.

[0015] Another compound of the invention consists essentially of theamino acid sequence depicted in Seq. ID No. 4 wherein the compoundpossesses at least one, and typically two, of the following properties:(1) it binds to a binding site on wild type p53 protein and (2) itcompetitively inhibits the binding of wild type p53 protein to wild typeXPB protein. Preferably, this compound consists of the amino acidsequence depicted in Seq ID No. 4. Another method of diagnosing aXeroderma pigmentosum complementation group, such as group B or D, in anindividual includes providing a sample cell derived from the individual,contacting the sample cell with a compound of the invention, anddetecting whether or not apoptosis of the sample cell occurs, therebydiagnosing whether or not the sample cell contains at least one mutantXeroderma pigmentosum gene such as a mutant XPB gene, a mutant XPD gene,or both.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0016]FIGS. 1A and 1B. Human wild-type and mutant p53 protein are ableto complex with XPB, XPD, CSR and Rad3 proteins in vitro. A. GSH-beadsloaded with either 4 μg of GST (lane 2) or 2 μl of GST-p53-WT (lane 3),GST-p53-135Y (lane 4), GST-p53-249S (lane 5), or GST-p53-273H (lane 6)were mixed with ³⁵S-labelled, in vitro-translated XPB (5 μl), XPD (5μl), Rad3 (5 μl), or CSB (15 μl). Proteins which remained bound wereanalyzed by SDS/PAGE. The authentic XPB, XPD, CSR and Rad3 proteinsimmunoprecipitated by the specific antibodies were loaded in lane 1 asreferences. In lane 1, the indicated fraction of XPB, XPD, Rad3, or CSBwas immunoprecipitated by anti-XPB polyclonal antibodies (Ab) as a 90kDa protein, MAb2F6 (anti-XPD monoclonal Ab) as an 80 kDa protein,anti-Rad3 polyclonal Abs as a 85 kDa protein, or anti-CSB polyclonal Absas a 170 kDa protein, respectively. GST-p53-WT, wild-type p53;GST-p53-135Y, p53 mutated at codon 135 (His-Tyr); GST-p53-249S, p53mutated at codon 249 (Arg→Ser); GST-p53-273H, p53 mutated at codon 273(Arg→His). B. The percentage of total protein bound was quantitated bydensitometry. The binding between experiments varies by less than 10percent.

[0017]FIGS. 2A and 2B. Carboxyl terminus of p53 is important forassociation with XPB, XPD, CSR and Rad3 proteins. A. In vitro translatedXPB, XPD, CSR and Rad3 proteins were incubated with GSH-beads loadedwith either 4 μg of GST (lane 2), or 2 μg each of GST-p53-WT (lane 3),or various GST-p53 deletion mutants (lanes 4-8). In lane 1, theindicated fraction of the XPB, XPD, CSR and Rad3 proteins wereimmunoprecipitated as in FIG. 1 for the references. B. Schematicrepresentation of wild-type and deletion mutants of human p53 proteinsand a summary of their binding properties with in vitro-translated XPB,XPD, CSR and Rad3 proteins. ΔC50, deletion of 50 residues at theC-terminus of p53; N5, deletion of 100 residues at the C-terminus ofp53; 2C, deletion of 94 residues at the N-terminus of p53, 3C, deletionof 155 residues at the N-terminus of p53; 25, deletion of both 94residues at the N-terminus and 100 residues at the C-terminus of p53.The ERCC protein binding site is indicated. The black boxes representthe evolutionarily conserved domains of p53. Degrees of binding: +,binding; +, reduced binding; −, no binding.

[0018]FIGS. 3A, 3B and 3C. p53 binds to XPB helicase motifs Ia, II andIII. A. ³⁵S-labeled, In vitro-translated wild-type XPB (lanes 1 and 4),deletion mutant ΔC496 (lanes 2 and 5), or deletion mutant ΔC354 (lanes 3and 6) was mixed with GSH-beads loaded with 2 μg of GST-p53-WT and boundproteins were analyzed on SDS/PAGE (lanes 4-6). Twenty percent of theoriginal input was loaded in parallel as the references (lanes 1-3). B.In vitro-translated wild-type p53 protein was mixed with GSH-beadsloaded with 4 μg of GST (lane 2), 2 μg of GST-XPB (WT) (lane 3), or 2 μgof GST-XPB (truncated XPB with 80-480 residues) (lane 4). Bound proteinswere analyzed on SDS/PAGE. The original input (100%) was included as areference (lane 1). C. Schematic representation of the full-length orthe truncated XPB proteins used in binding assays. Black boxes in thebars are the putative helicase motifs conserved across the helicasesuperfamily. The small open bar from residues 354 to 496 covers theregion which binds p53, with the closed bar in the right side coveringmotif III representing the higher affinity binding site. Helicase motif1, residues 337-351; motif Ia, residues 361-374; motif II, residues434-446; motif III, residues 462-480; motif IV, residues 576-592; motifV, residues 601-621; motif VI, residues 630-649.

[0019]FIG. 4. Peptides corresponding to helicase motif III of XPB andthe C-terminus of p53 prevent XPB from binding to GST-p53. Fourdifferent synthetic peptides were pre-incubated with 2 μg GST-p53WT for30 minutes on ice before the addition of ³⁵S-labeled, invitro-translated XPB for 60 min at RT. Peptide #464 corresponds toresidues 464-478 of XPB (lanes 2-4; 12, 120, and 596 nM), peptide #479corresponds to residues 479-493 of XPB (lanes 5-7; 12, 116, and 578 nM),peptide #99 corresponds to residues 100-115 of HBX (lanes 8-9; 111 and554 nM), and peptide #p53cp corresponds to residues 367-387 of p53(lanes 11-12; 85 and 424 nM).

[0020]FIGS. 5A, 5B and 5C. Wild-type but not mutant p53 inhibitshelicase activity of XPD (Rad3 homologue) and XPB, the major componentsof BTF2-TFIIH transcription/repair factor. A. Substrate is indicated onthe right; the 27 nt (top band) is displaced by XPD (5′-3′ helicase) andthe 24 nt (bottom band) is displaced by XPB (3′-5′ helicase). The³²P-labeled substrate was incubated with highly purified BTF2-TFIIH (HAPfraction) in the absence (lanes 3 and 4) and in the presence of 6, 18,and 120 ng of baculovirus-produced wild-type p53 (lanes 5-7), or 7, 21,and 140 ng of mutant p53-273H (lanes 8-10). In lane 1, the substrate washeated for 2 min at 100° C. (CΔ) and in lane 2, the substrate was loadeddirectly (−). Pichia pastoris-produced wild-type p53 produced identicalresults as in lanes 5-7. B. Effect of p53 on Rad3 helicase activity. DNAhelicase activity was measured as described (49) using 90 ng of Rad3protein in the absence (lane 3) or presence of 50 ng or 200 ng of p53WT(lanes 1 and 2), or 200 ng of mutant p53-273H(B) (lane 4). Helicaseactivity of 90 ng of Rad3 protein was assayed without 1 mM ATP (lane 5).C. Inhibition of XPD and XPB helicase activities by wild-type p53. Thequantitative results obtained from densitometry analysis of theautoradiography shown in FIG. 6A with the amounts of oligomers displayedby XPB and XPD proteins and expressed in the helicase activity as afunction of p53 doses. O, XPD+WT; Δ, XPB+WT; , XPD+273H; ▴, XPB+273H.

[0021]FIG. 6. No inhibition of the BTF2-TFIIH-associated invitro-transcription of RNA polymerase II or ATPase activity by p53. Thepurified BTF2-TFIIH was preincubated for 20 minutes at 4° C. without(lane 1) or with 6, 18, and 120 ng of baculovirus—produced wild-type p53(lanes 2-4), or 7, 21, and 140 ng of mutant p53-273H (lanes 5-7). Thetranscription reaction was then completed by addition of RNA polymeraseII, DNA template, nucleotides and the other basal transcription factorsincluding either yeast TBP (yTBP) or human TFIID (hTBP), and byincubation for 45 min at 25° C. The transcripts were analyzed asdescribed in Example 5, herein. The specific transcript of a 309nucleotide long (nt) either by yTBP or hTBP is indicated. The ATPaseactivity of the BTF2-TFIIH with or without p53 was measured as describedin Materials and Methods. Pi, inorganic phosphate that is liberated fromATP.

[0022]FIGS. 7A, 7B, 7C, 7D, 7E and 7F. Induction of apoptosis bymicroinjection of the wild-type and various mutant p53 expressionvectors in normal primary human fibroblasts. Cells were injected withthe expression vectors, including wt (A, B), 143^(ala) (C, D), and249^(ser) (E, F), and were incubated for 24 hr prior to fixation. p53protein was stained with CM-1 antibody (A, C, E). Nuclei were stained byDAPI (B, D, F).

[0023]FIG. 8. Differential induction of apoptosis between normal primaryhuman fibroblasts and primary fibroblasts from Xeroderma pigmentosum(XP-B and XP-D) donors following microinjection of the wild-type p53expression vector.

DETAILED DESCRIPTION

[0024] The present invention provides novel methods for screening largenumbers of test compounds for those which have the desirable property ofinducing apoptosis and which are therefore candidate compounds fortreatment of human cancers. There are a variety of agents, includinghigh concentrations of wild type p53 protein, which are capable ofinducing certain cells to undergo apoptosis.

[0025] High concentrations of wild type (wt) p53 protein can induceapoptosis in a variety of different tumor cells. Tumor cells that aresusceptible to induction of apoptosis by wild type p53 proteins includethose tumor cells which have a mutant p53 protein. The term “mutant p53protein”, as used herein, refers to mutations in the p53 gene whichalter expression of p53 protein in the cell or which result in theproduction of a p53 protein which is structurally different that wildtype p53 protein. Mutant p53 proteins may result from point mutations ordeletion mutations in the p53 gene. A variety of different p53 mutationshave been identified which are associated with a number of differentmalignancies. See Hollstein, M. et al. (1991) Science, 253:49-53, for adescription of p53 mutations found in number of different cancers.

[0026] The present invention relies, in part, on the discovery that thep53-dependent apoptosis pathway involves an interaction of p53 proteinwith XPB and/or XPD proteins. XPB and XPD proteins form part of the RNApolymerase II basal transcription factor, TFIIH. TFIIH contains at leastfive subunits including XPB, XPD, p62, p44, and p34. XPD and XPB bothpossess helicase activity and are indispensable for nucleotide excisionrepair (NER). See Schaeffer, L., et al., EMBO Journal (1994)13:2388-2392 and Schaeffer, L., et al., (1993) Science 260:58-63, for adescription of XPD and XPB proteins and of TFIIH. As demonstrated inExamples 2-8, herein, p53-dependent apoptosis is mediated, at least inpart, by the binding of wild type p53 protein to XPB and XPD proteins.Furthermore, as demonstrated in Example 6, herein, the binding of p53protein to XPD or XPB proteins results in inhibition of the helicaseactivity of these proteins.

[0027] The present invention encompasses a variety of screening assaysfor compounds that are capable of inducing apoptosis in tumor cells, andwhich are based on the interaction between p53 protein and XPB or XPDproteins. The terms “screening assay for apoptosis” or “methods forscreening a compound for the ability to induce apoptosis”, as usedherein, refer to assay methods which are capable of detecting compoundsthat have the biological activity of inducing apoptosis in certaincells, such as tumor cells having either a wild type or mutant p53protein.

[0028] The screening assays of the invention can be used for screeninglarge numbers of compounds to identify a group of compounds that arecandidate compounds for clinical use for treatment of certain cancers.Other compounds that do not have activity in the screening assays can beeliminated from further consideration as candidate compounds. Thescreening assays therefore have utility in the pharmaceutical industry.In addition to identifying candidate compounds for treatment ofmalignant diseases, the screening assays are also useful for identifyingcompounds that can be used in in vitro diagnostics, for example, in thediagnosis of Xeroderma pigmentosum (see below).

[0029] There are a variety of different assays for detecting compoundscapable of inducing apoptosis that are encompassed by the presentinvention. For example, there are a number of different screening assaysthat are based on the binding of wild type p53 protein to XPB and/or XPDproteins. For instance, compounds which inhibit the binding of p53protein to XPD or XPB protein can be identified in competitive bindingassays. Alternatively, the binding of a test compound to XPB or XPDprotein can be measured directly, in the presence or absence of wildtype p53 protein. This latter type of assay is called a direct bindingassay. Both direct binding assays and competitive binding assays can beused in a variety of different formats, similar to the formats used inimmunoassays and receptor binding assays. For a description of differentformats for binding assays, including competitive binding assays anddirect binding assays, see Basic and Clinical Immunology 7th Edition (D.Stites and A. Terr ed.) 1991; Enzyme Immunoassay, E. T. Maggio, ed., CRCPress, Boca Raton, Fla. (1980); and “Practice and Theory of EnzymeImmunoassays,” P. Tijssen, Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers B. V. Amsterdam (1985),each of which is incorporated herein by reference.

[0030] In competitive binding assays, for example, the sample compoundcan compete with a labeled analyte for specific binding sites on abinding agent bound to a solid surface. In this type of format, thelabeled analyte can be labeled p53 protein and the binding agent can beXPB or XPD protein bound to a solid phase. Alternatively, the labeledanalyte can be labeled XPB and/or XPD protein and the binding agent canbe a solid phase wild type p53 protein. The concentration of labeledanalyte bound to the capture agent is inversely proportional to theability of a test compound to compete in the binding assay. An exampleof a competitive binding assay for detecting compounds capable ofinhibiting the binding of wild type p53 protein to XPD or XPB protein isdescribed in Example 5, herein. The amount of inhibition of labeledanalyte by the test compound depends on the binding assay conditions andon the concentrations of binding agent, labeled analyte, and testcompound that are used. Under specified assay conditions, a compound issaid to be capable of inhibiting the binding of p53 protein to XPB orXPD protein in a competitive binding assay, if the amount of binding ofthe labeled analyte to the binding agent is decreased by 10% or more.When a direct binding assay format is used, a test compound is said toinhibit the binding of p53 protein to XPB or XPD protein when the signalmeasured is twice the background level or higher.

[0031] In a competitive binding assay, the sample compound competes withlabeled protein for binding to a specific binding agent. As describedabove, the binding agent may be bound to a solid surface to effectseparation of bound labelled protein from the unbound labelled protein.Alternately, the competitive binding assay may be conducted in liquidphase, and any of a variety of techniques known in the art may be usedto separate the bound labeled protein from the unbound labeled protein.Following separation, the amount of bound labeled protein is determined.The amount of protein present in the sample is inversely proportional tothe amount of labelled protein binding.

[0032] Alternatively, a homogenous binding assay may be performed inwhich a separation step is not needed. In these type of binding assays,the label on the protein is altered by the binding of the protein to itsspecific binding agent. This alteration in the labelled protein resultsin a decrease or increase in the signal emitted by label, so thatmeasurement of the label at the end of the binding assay allows fordetection or quantitation of the protein.

[0033] The binding assay formats described herein employ labeled assaycomponents. The label can be in a variety of forms. The label may becoupled directly or indirectly to the desired component of the assayaccording to methods well known in the art. A wide variety of labels maybe used. The component may be labeled by any one of several methods.Traditionally, a radioactive label incorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P is used. Non-radioactive labels include ligands which bind tolabeled antibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabelled ligand. The choice of label depends on sensitivity required,ease of conjugation with the compound, stability requirements, andavailable instrumentation. For a review of various labelling or signalproducing systems which may be used, see U.S. Pat. No. 4,391,904, whichis incorporated herein by reference.

[0034] The methods of the invention also encompass the use ofbiologically active fragments of p53 protein or XPB or XPD proteins inthe screening assays of the invention. The term “biologically activefragment of wild type p53 protein” refers to fragments of the p53protein that bind to XPB or XPD proteins. The terms “biologically activefragment of XPB protein” and “biologically active fragment of XPDprotein” refer to those fragments of XPB protein and XPD proteinrespectively that bind to wild type p53 protein. Methods of productionof wild type p53 protein and wild type XPD and XPB proteins, for use inscreening assays are known to those of skill in the art. For example,these proteins may be produced as recombinant proteins as described inExample 1, herein.

[0035] Another type of screening assay encompassed by the presentinvention is an assay which identifies compounds capable of inhibitingthe helicase activity of XPD or XPB protein. In this type of assay, testcompounds are incubated with XPD and/or XPB protein, and the helicaseactivity is determined. The helicase activity can then be compared to acontrol which lacks the test compound.

[0036] XPD or XPB proteins with helicase activity can be present in thescreening assay as individual proteins or as a part of the TFIIHtranscription factor complex. TFIIH transcription factor complex can bepurified from a variety of different sources by methods known by one ofskill in the art. For example, the TFIIH complex can be isolated asdescribed in Example 6, herein. XPD or XPB proteins can also be isolatedfrom natural sources or can be produced as recombinant proteins, forinstance, as described in Example 1, herein. See Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold SpringHarbor Laboratory, (1989) or Current Protocols in Molecular Biology, F.Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York(1987) for a description of techniques useful in the production ofrecombinant proteins.

[0037] Helicase activity can be determined by a variety of assays knownto those of skill in the art. For example, helicase activity can bedetermined as described in Schaeffer, L., et al. (1993) Science260:58-63, 1993; Schaeffer, L., et al. (1994) EMBO J. 13:2388-2392; andRoy, R., et al. (1994) J. Biol. Chem 269:9826-9832, and in Example 6,herein. When XPB and XPD are present in the TFIIH complex, the helicaseactivity of XPB, which initiates DNA unwinding in the 3′ to 5′direction, and of XPD, which initiates DNA unwinding in the 5′ to 3′direction can both be determined as described in Example 6 herein.

[0038] A compound capable of inhibiting the helicase activity of XPB orXPD protein is a compound that inhibits at least 10% of the helicaseactivity of XPB or XPD protein in such an assay. Preferably, thecompound inhibits at least 50% of the helicase activity of XPB or XPDprotein, and more preferably, the compound inhibits at least 80% of thehelicase activity of XPB or XPD protein.

[0039] Screening assays utilizing cells expressing mutant XPB and/ormutant XPD proteins are also encompassed by the present invention. Thesetypes of screening assays are useful for detecting compounds that arecapable of inducing apoptosis by a pathway involving XPD or XPBproteins. At least two different types of cells with differing geneticcompositions are used, typically in separate cultures, in this type ofassay. One type of cell contains either a wild type or mutant p53 gene,as defined herein, and is capable of undergoing apoptosis when a DNAconstruct expressing wild type p53 protein is introduced into the cell.This cell also contains XPB and XPD proteins that are functionally wildtype. The terms “wild type XPB” and “wild type XPD”, as used herein,refer to those proteins that have helicase activity, which arefunctional when present in normal TFIIH complexes, and that are capableof binding wild type p53 protein.

[0040] A second type of cell that is typically tested in a parallelculture in the cellular screening assay is a cell that has a mutant XPBprotein and/or a mutant XPD protein, and which is less capable ofundergoing apoptosis after introduction of a DNA construct expressingwild type p53. The terms “mutant XPB gene” or “mutant XPB protein” referto those XPB mutations which confer on a cell the phenotype of having areduced amount of apoptosis when DNA expressing wild type p53 isintroduced into the cell. The terms “mutant XPD gene” or “mutant XPDprotein” refer to those XPD mutations which confer on a cell line thephenotype of having a reduced amount of apoptosis when DNA expressingwild type p53 is introduced into the cell line. Cells with mutant XPBand/or mutant XPD proteins may still be able to undergo some degree ofapoptosis, but the amount is diminished as compared to control cells.

[0041] Cells expressing mutant XPD and/or mutant XPB can be prepared ina variety of ways. For example, such cells can be isolated from patientswith genetic disorders affecting the XPD or XPB genes such as Xerodermapigmentosum. See Example 8, herein, for examples of cells with mutantXPB or mutant XPD genes, which are useful in the screening assays of theinvention.

[0042] Test compounds are introduced into cell cultures of the cell typemore capable of undergoing apoptosis when DNA expressing wild type p53is put into the cell. Test compounds are also introduced into the cellcultures of a cell type containing mutant XPD and/or mutant XPB.Apoptosis is measured and compounds that are capable of inducingapoptosis it the first cell type, but that are less capable of inducingapoptosis in the second cell type, are selected.

[0043] Apoptosis can be measured by a variety of techniques. Forexample, apoptosis can be measured by determination of cell phenotype.Phenotype refers to how the cell looks, typically microscopically, butgross or macroscopic appearance can be observed. The phenotype changesdepending on the growth rate of the cells. For instance, the microscopicmorphology of cells that are rapidly dividing and growing is differentthan that of cells undergoing cell death by apoptosis. Determination ofcell phenotype is well within the ability of one with ordinary skill inthe art.

[0044] There are also a number of biochemical assays that can be used todetect apoptosis, such as “laddering” of the cellular DNA. When testingcompounds for the ability to induce apoptosis, cell death (notcytostasis) is an endpoint of compound application to the cell. Aclassic signature of apoptosis is the cleavage of nuclear DNA intonucleosomal subunits. On gels, this gives rise to the appearance of aladder as nucleosomal units are sequentially cleaved from the DNA.Observation of a classic DNA ladder is indicative of apoptosis. Forexample, cells are lysed and the high molecular DNA is removed bycentrifugation. The aqueous phase is treated with proteinase K to digestproteins. After a phenol/chloroform extraction, the DNA is precipitatedwith salt and ethanol. The pellet is dissolved in deionized water andtreated with 500 μg/ml RNase A. The DNA is run on a 2% agarose minigel.Observation for a classic DNA ladders is made. A gel photograph can betaken. Cell death is verified by the demonstration of DNA fragmentationas represented by the ladder configurations on the gel. (See Gavrieli,Y., et al. (1992) J. Cell Biol. 119:493). There are also a variety ofother assays available for apoptosis such as “TUNEL” assays (see White,E., et al. (1984) J. Virol. 52:410). See also Example 7, herein, for ademonstration of the determination of apoptosis.

[0045] More than two types of cells can be used in the cellular assaydescribed above. For example, multiple cell lines containing a mutantp53 and/or containing wild type p53 could be used. Multiple cultures ofcells with different mutant XPD or mutant XPB protein may also beuseful. Furthermore, a variety of different cell lines or cells ofdifferent origin can be used. See examples 7 and 8, herein, for ademonstration of the use of human fibroblastic cells and for an exampleof assay conditions that can be adapted for screening test compounds.

[0046] Other types of screening assays based on the interaction of p53protein with XPB and/or XPD proteins are also encompassed by theinvention. In addition, the different types of screening assaysdescribed herein may be used in combination with each other to increasethe usefulness of the assays. For example, test compounds could first bescreened in a binding assay to detect compounds which bind to XPD orXPB. Compounds having binding activity could then be tested in anotherscreening assay, such as the cellular assay described above which usescells with XPD or XPB mutants.

[0047] The present invention also provides compounds that are active inone of the above-described screening assays. These compounds are capableof inducing apoptosis in cells that are susceptible to p53-mediatedapoptosis, such as tumor cells. These compounds can also be capable ofaffecting the interaction of p53 protein with XPB and/or XPD proteinsand/or the helicase activity of the XPD or XPB proteins.

[0048] The compounds of the invention include peptides from the p53amino acid sequence that are capable of blocking the binding of p53protein to XPB protein or the binding of p53 protein to XPD protein.With regard to p53 protein, the full length amino acid sequence of humanwild type p53 is shown in Seq. ID No. 1. As described in Example 3,herein, the C-terminal domain of p53 protein contains the binding regionfor binding to XPB protein. Furthermore, as described in Example 5herein, peptide #p53cp from the C-terminal region of p53 protein iscapable of inhibiting the binding of wild type p53 protein to XPBprotein. Peptide #p53cp is depicted in Seq. ID No. 2 and consists ofamino acid residues 367-387 of human wild type p53.

[0049] The compounds of the invention also include peptides from the XPBand XPD amino acid sequences that are capable of inhibiting the bindingof p53 protein to XPB protein or XPD protein, respectively. With regardto XPB protein, the entire amino acid sequence of human XPB protein isshown in Seq. ID No. 3. As described in Example 4, herein, the bindingregion of XPB protein for p53 protein is located in the helicase motifIII region of XPB protein. Furthermore, as described in Example 5herein, peptide #464 from the helicase III region of XPB protein iscapable of inhibiting the binding of wild type p53 protein to XPBprotein. Peptide #464 is depicted in Seq. ID No. 4 and consists of aminoacid residues 464 to 478 of human wild type p53.

[0050] Peptide compositions of the invention include not only thespecific peptide sequences shown in Seq. ID Nos. 2 and 4, but alsofragments of these two peptides that retain the ability to block thebinding of p53 protein to XPB protein. The peptides of Seq. ID No. 2 andSeq. ID No. 4 may also have non-essential moieties attached to thepeptides. The term “non-essential moieties”, as used herein, refers tothose chemical moieties that do not prevent the peptide from inhibitingthe binding of p53 protein to XPB protein or to XPD protein. Forexample, the term “non-essential moieties” includes amino acid sequenceextensions at either the amino-terminal or carboxy-terminal end ofpeptides of Seq. ID Nos. 2 and 4 which do not prevent these peptidesfrom inhibiting the binding to p53 protein to XPB protein or to XPDprotein. Examples of such amino acid sequence extensions include thenaturally occurring amino acid sequences of p53 protein and XPB proteinthat are depicted in Seq. ID Nos. 1 and 3, respectively. Preferably,such amino acid extensions are no longer than 100 amino acids in lengthat either that C-terminal or amino terminal end of peptides of Seq. IDNos. 2 or 4, and more preferably are no longer than 50 amino acids inlength. The ability of any such peptides to block helicase activity ofXPB or XPD protein can be determined by the methods described herein.

[0051] The peptides of the invention may also have conservative aminoacid substitutions from the sequences depicted in Seq. ID Nos. 2 and 4.The substitution of amino acids having similar chemical properties suchas charge or polarity are not likely to affect the ability of thepeptides to inhibit the binding of p53 protein to XPB protein. Examplesinclude substitutions of asparagine for glutamine or aspartic acid forglutamic acid.

[0052] Peptide compositions of the invention can be produced by avariety of ways known to those of skill in the art. For example, thepolypeptides of the invention can be synthetically prepared by widevariety of methods. For instance, polypeptides of relatively short sizecan be synthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed.,Pierce Chemical Co. (1984). Polypeptides of the invention can also beprepared using recombinant DNA technology. See Sambrook, et al., supra.

[0053] Peptides of the invention can be modified according to standardtechniques to yield compounds with a variety of desired properties. Forinstance, the polypeptides can vary from the naturally-occurringsequences described herein at the primary structure level by amino acidinsertions, substitutions, deletions, and the like. The amino acidsequence variants can be prepared with various objectives in mind,including facilitating purification and preparation of recombinantpolypeptides. Such modifications can also be useful in, for example,modifying plasma half-life, improving therapeutic efficacy, andlessening the severity or occurrence of side effects during therapeuticuse.

[0054] The invention provides methods of diagnosing Xerodermapigmentosum complementation group B or D in an individual. A positivediagnosis confirms that the individual has a genetic basis for one orboth of the graver forms of the disease. To perform the method, thepractitioner selects an individual suspected of having a XP mutation.Testing may include fetal testing in pregnant women. The suspicion canbe based on clinical findings, symptoms, family history, or laboratorystudies.

[0055] A cellular sample is taken from the individual. This sampleincludes cellular material, and preferably includes whole cells. Thesample can employed fresh, but it is usually grown in tissue culturebefore performing the test. Consequently, the sample can be any cellularmaterial derived from the individual or from a fetal sample. The samplecan be taken by any of a number of techniques including tissue scraping,biopsy, washings and aspiration. For example, the clinician can take amucous membrane scraping, typically buccal or cervical; a skin biopsy;or a bone marrow aspiration. Preferably, skin fibroblasts or bloodlymphocytes are used.

[0056] The sample cell is contacted with a compound of the invention,preferably the peptide of Seq. ID No. 2 or Seq. ID No.4. Contact ispreferably made by applying the compound directly in an aqueous solutionto the sample in a tissue growth medium. Alternatively, a DNA constructexpressing the compound could be prepared and microinjected into thecell(s).

[0057] After a sufficient time period, the practitioner observes whetheror not apoptosis of the material derived from the cellular sampleoccurs. Detection of apoptosis can be accomplished by any of a number ofmeans such as those discussed herein. For example, preparation andstaining for DNA ladders is done, although other methods, direct andindirect, are available. If apoptosis is detected, then the practitionerdiagnoses that the sample cell contains at least one mutant Xerodermapigmentosum gene, either a mutant XPB gene or a mutant XPD gene, orboth.

[0058] The foregoing description and the following examples are offeredprimarily for purposes of illustration. It will be readily apparent tothose skilled in the art that the operating conditions, materials,procedural steps and other parameters of the system described herein maybe further modified or substituted in various ways without departingfrom the spirit and scope of the invention. Thus the invention is notlimited by the description and examples, but rather by the appendedclaims.

EXAMPLES Example 1

[0059] Production of Recombinant Proteins:

[0060] A. Production of Expression Plasmids:

[0061] GST-p53-WT encodes glutathione S-transferase (GST) fused to humanwild type p53. GST-p53-135Y, -249S, and -273H encode GST fused to p53mutated at codon 135 (his-tyr), 249 (arg-ser), and 273 (arg-his),respectively. The production of these constructs are described in Wang,X. W., et al. (1994) Proc. Natl. Acad. Sci. USA 91:2230-2234. ΔC50encodes a GST-p53 fusion protein with a deletion of the last 50 aminoacids. ΔC50 was made by PCR amplification of the first 1,028 nucleotides(nt) of p53 from pC53-SN, (obtained from of Bert Vogelstein, JohnsHopkins University. Korn, S. E., Pietenpol, J. A., Thiragalingam, S.,Seymour, A., Kirzler, K. W. and Vogelstein, B., Science 256:827-830,1992. The PCR product was inserted into the BamHI site of pGEX-2T(Pharmacia LKB, Uppsala, Sweden). Plasmids N5, 2C, 3C, and 25 asdescribed in Ruppert, J. M. and Stillman, B., (1993) Mol. Cell Biol.13:3811-3820 encode GST fused to amino acids 1-293, 94-393, 155-393, and94-293 of p53, respectively. These plasmids were obtained from BruceSpillman at Cold Spring Harbor Laboratory. Plasmid pZAP10, encoding aXPB cDNA under the control of bacterial T7 promoter, was used for invitro translation of full length XPB protein, and was linearized withPstI and SphI for in vitro translation Of ΔC496 and ΔC354.

[0062] pGEM3zf(+)T7XPD was used for in vitro translation of XPD proteinwith a T7 promoter. pGEM4z-SP6ccaccRAD3 was used for in vitrotranslation of Rad3 protein with a SP6 promoter. pcBLsSE6 was used forin vitro translation of CSB with a T7 promoter. GST-XPB-80/480 encodesGST fused to amino acids 80 to 480 of XPB. GST-XPB, encoding GST fusedto full length XPB, was constructed by PCR amplification of the XPBcoding region in pZAP10 and insertion into the BamHI and EcoRI sites ofpGEX-2T. pSelectp53 was used for in vitro-translation of human wild-typep53 protein as described in Wang, X. W., et al. (1994) Proc. Natl. Acad.Sci. USA 91:2230-2234. pPOLY(A)-luc (SP6) (Promega, Madison, Wis., USA)was used for in vitro translation of luciferase. pcref, constructed byChris Chay in the Laboratory of Human Carcinogenesis, National CancerInstitute, Bethesda, Md. 20892, was used for in vitro translation ofREF1.

[0063] B. Expression and Purification of Recombinant Proteins:

[0064] GST fusion proteins were produced in Escherichia coli andpurified on glutathione-Sepharose 4B beads (GSH-beads) according to themanufacturers' directions (Pharmacia LKB). The purified fusion proteinsimmobilized on the surface of GSH-beads were stored at 4° C. inphosphate-buffered saline, pH7.4, containing 1% Triton X-100 for up totwo months. Protein concentrations were determined by Coomassie bluestaining of SDS/PAGE and comparison to molecular weight standards(BioRad, Hercules, Calif., USA) run on the same gel. Highly purifiedbaculovirus-produced p53-WT and p53-273H proteins were kindly providedby Carol Prives (Columbia University), Wang E. H, Friedman, P. N. andPrives, C., Cell 57:379-392, 1989, and were proved to be thebiologically active forms. To label the in vitro translated proteins,the corresponding plasmids were used in a one-step in vitrotranscription and translation system (Promega) at room temperature (RT)for 90 min in the presence of [³⁵S]Cysteine (Dupont, Boston, Mass.,USA). In vitro-translated proteins were freshly prepared each time,prior to use.

Example 2

[0065] Binding of Wild Type and Mutant p53 With Nucleotide ExcisionRepair (NER) Proteins

[0066] A. Binding of Wild Type p53

[0067] In order to demonstrate that wild type p53 interacts with NERproteins, human wild type p53 protein was tagged at the N-terminus withglutathione S-transferase (GST) as described in Example 1. p53 proteinwas incubated with in vitro translated ³⁵S-labeled XPD, Rad3, XPB, orCSB protein, which were also produced as described in Example 1. Thebinding assays were done in 500 μl IP buffer (50 mM Tris-HCl, pH8.0/120mM NaCl/0.5% Nonidet P-40) containing the ³⁵S-labeled, in vitrotranslated proteins and GST fusion proteins loaded on GSH-beads at RTfor 60 min. After washing five times with IP buffer, the bound proteinswere released by boiling the beads in Laemmli buffer for 5 min,separated by SDS-PAGE, and visualized by flurography, as described inWang, X. W., et al. (1994) Proc. Natl. Acad. Sci., USA 91:2230-2234. Theresults are shown in FIG. 1. XPD, XPB and CSB bind to GST-p53-WT with arelatively higher affinity (about 20% of input) than Rad3 (about 10% ofinput) (see FIG. 1A, comparing lane 1 with 3). GST alone did notinteract with any of the four proteins (lane 2, FIG. 1). Invitro-translated REF1, a nuclear factor involved in DNA repair and aredox pathway, as well as in vitro-translated luciferase and othernon-relevant proteins present in the translation mix, did not bind toeither GST-p53-WT or GST-XPB. Binding was not mediated through singlestranded (ss) DNA and RNA, as was demonstrated by the treatment of invitro-translated products with DNAse and RNAse prior to binding.

[0068] B. Binding of Mutant p53 Proteins

[0069] In order to demonstrate the binding of several mutant p53proteins to NER proteins, equal amounts of GST-tagged p53 mutants,(135Y, 249S, 273H) were tested for binding to the in vitro-translatedXPD, XPB, CSB and Rad3 proteins used above. The mutant p53 proteins wereproduced as described in Example 1 and were tested for binding to NERproteins in the same manner as was wild type p53. The results are shownin FIG. 1. All the mutants tested had similar or increased binding tothe human proteins but decreased binding to yeast Rad3, as compared toGST-p53-WT (see FIG. 1A, comparing lane 3 with lanes 4, 5, and 6). Whilenot wishing to be bound by theory, this opens the possibility thatmutant p53 may exert a dominant negative effect by binding to andsequestering the cellular targets of wild-type p53. The p53-135Y mutant,which has diminished binding to hepatitis B virus X protein andpapillomavirus E6 protein, binds to XPD, XPB, Rad3 and CSB. HBX has alsobeen shown to inhibit p53 binding to XPB presumably by binding to thesame site of p53 that interacts with XPB, since HBX did not bind to XPBin vitro (see Wang, X. W., et al. (1994) Proc. Natl. Acad. Sci., USA91:2230-2234.)

Example 3

[0070] Binding of Mutant p53 Proteins with C-terminal Deletions toNucleotide Excision Repair (NER) Proteins

[0071] Defining the protein domains required for various interactionscan provide clues to the significance of the interaction, especially ifthe domains are functionally important. The importance of the C-terminaldomain of p53 for binding to NER proteins was demonstrated by the use ofmutant p53 proteins with C-terminal deletions that were tagged with GST.The deletion mutant p53-GST fusion proteins were prepared as describedin Example 1 herein, and binding studies to NER proteins were performedas described in Example 2 herein. The results are shown in FIG. 2.

[0072] Deletion of the 50 C-terminal amino acids abolished binding ofXPB, Rad3 or CSB, and reduced binding of XPD, while deletion of up to155 N-terminal amino acids had no detectable effect (see FIG. 2). XPDbinding diminished with deletion of the 100 C-terminal amino acids (FIG.2A, comparing lane 5 to lane 3). The effect of C-terminal deletions ofvarious length on p53 binding to NER proteins is summarized in FIG.2(C). The results suggest that the C-terminal region of p53 is involvedthe binding of p53 to XPD and XPB.

Example 4

[0073] Binding of XPB Proteins With Deletion Mutations to Wild Type p53:

[0074] XPB contains 7 putative helicase motifs (FIG. 3C, black bars)which are conserved among the helicase superfamily and are indispensablefor NER activity. In order to demonstrate that p53 interacts with ahelicase domain of XPB, three C-terminal deletion mutants of XPB weretranslated in vitro and assayed for binding to GST-p53-WT. The XPBmutants were produced as described in Example 1, herein, and the bindingstudies with wild type p53 were carried out as described in Example 2,herein. The results are shown in FIG. 3.

[0075] A deletion mutant terminating at residue 496 (mutant ΔC496, i.e.,deletion of helicase motifs IV, V and VI) did not alter the binding towild type p53 (see FIG. 3A, comparing lane 5 to lane 2). However,further deletion to residue 354 (mutant ΔC354, i.e., deletion ofhelicase motifs Ia, II, III, IV, V and VI) completely abolished thebinding to p53 (see FIG. 3A, comparing lane 6 to lane 3). Whilefull-length GST-XPB fusion protein effectively binds in vitro-translatedhuman wild-type p53 (FIG. 3B, compare lane 3 to lane 1), an XPB fragmentcontaining residues 80-480 tagged with GST showed significantly lessbinding (FIG. 3B, compare lane 4 to lane 3). Similar patterns wereobserved when GST-p53-135Y, 249S or 273H mutants were used (data notshown). These results indicate that p53 binds to XPB at a region withinor near helicase motif III (residues 462-495).

[0076] We used the Chou-Fasman and the Robson-Garnier methods (see Chou,P. Y. and Fasman, G. D., (1974) Biochemistry 13:222-245, and Garnier,J., et al. (1978) J. Mol. Biol. 120:97-120) to predict possiblesecondary structures for the two interacting regions of p53 and XPB. Theconserved helicase motif III consists of a 3-6 residue turn containing1-3 acidic (negatively charged) residues, which are likely to be exposedon the protein's surface, and which are separated by two α-helices orβ-sheets, depending on the particular member of the superfamily.Sequence analysis of the C-terminal p53 domain reveals that it containsa stretch of basic (positively charged) amino acids (residues 367 to387) which are evolutionarily conserved from Xenopus to human. Thisregion is likely to form an a-helix with all the positively chargedresidues facing one side. While not wishing to be bound by theory, wehypothesize that this positively charged a-helical domain of p53 may bein direct contact with the negatively charged turn of helicase motif IIIin XPB.

[0077] The p53 tumor suppressor gene product selectively binds toseveral helicase proteins which are part of a transcription/repaircomplex, BTF2-TFIIH. This conclusion is based on the observations that:a) GST-p53-WT binds specifically to in vitro-translated XPD, XPB, CSB,or Rad3 proteins, but not to truncated XPB protein with helicase motifsIa, II, III deleted, and not to in vitro-translated luciferase and REFIproteins or to other non-relevant peptides present in the translationmix; b) full length GST-YPB binds to in vitro-translated p53 but aGST-XPB fragment (residues 80-480) has reduced binding activity; c)GST-p53-135Y mutant protein binds to the tested ERCC proteins althoughit has diminished binding to in vitro-translated hepatitis B viral Xprotein and human papillomavirus E6 protein; and d) GST-p53 deletionmutants have diverse binding activity to the XPD, XPB, CSB proteins.While not wishing to be bound by theory, the fact that p53 binds to manyhelicases which are involved in both RNA polymerase II transcription andNER suggests that p53 may play an important role in the maintenance ofgenomic integrity not only by controlling the cell cycle, but also bydirect participation in the nucleotide extension repair (NER) pathway.

Example 5

[0078] Inhibition of Binding of Wild Type 53 to XPB by p53 and XPBPeptides

[0079] The helicase motif III in XPB and the C-terminal region of p53are both important regions that are involved in the binding of p53 toXPB. We therefore used peptides corresponding either to helicase motifIII or to the p53 C-terminal domain to competitively inhibit the bindingof p53 to XPB. For these competitive binding studies, binding assayswere done by preincubation of GST-p53-WT containing beads with variousconcentrations of peptides for 30 min on ice followed by addition of invitro-translated proteins. Incubation conditions for the binding assaysand analysis of binding was then carried out as described in Example 2herein. The results are shown in FIG. 4.

[0080] Peptide #464 (residues 464 to 478 of XPB), depicted in Seq. IDNo. 4, efficiently competed in vitro-translated XPB from GST-p53 (seeFIG. 4, comparing lane 4 to lane 1), while peptide #479 (residues 479 to493 of XPB), depicted in Seq. ID No. 5, and a non relevant peptide #99(from HBV), depicted in Seq. ID No. 6, failed to do so. Instead, thelatter two peptides served to enhance binding (see FIG. 4, comparinglanes 6,7 and 9 to lane 1). We have no explanation why peptide #479 and#99 enhance binding. As expected, peptide #p53cp (residues 367 to 387 ofp53), and depicted in Seq. ID No. 2, also competed in vitro translatedXPB from GST-p53 (see FIG. 4A, comparing lane 12 to lane 10). Weconclude that motif III of XPB directly interacts with the p53C-terminal domain. However, these peptides did not compete XPD, CSB andRad3 from GST-p53 (data not shown). This may reflect a heterogeneityamong these helicases involved in the interaction. A more precisemolecular genetic approach, such as site-directed mutagenesis, will beneeded to further characterize these interactions. In addition, peptide#464 did not inhibit XPB from binding to mutant p53 (data not shown),suggesting that mutant p53 may require an additional binding site forXPB.

Example 6

[0081] Inhibition of the Helicase Activity of BTF2-TFIIH by Wild Typep53

[0082] We demonstrate below that p53 binding alters the helicaseactivity of XPD and XPB within the functional BTF2-TFIIH complex and theRad3 protein. Native BTF2-TFIIH was purified from a HeLa cell nuclearextract as described by Schaeffer, L., et al. (1993) Science 260:58-63.The helicase and ATPase assays of BTF2-TFIIH are essential as describedin Schaeffer, L., et al. (1993) Science 260:58-63, 1993; Schaeffer, L.,et al. (1994) EMBO J. 13:2388-2392; and Roy, R., et al. (1994) J. Biol.Chem 269:9826-9832. Purification of Rad3 as well as its ATPase andhelicase assays were as described elsewhere in Naegeli, H. et al. (1992)J. Biol. Chem 267:392-398. The BTF2-TFIIH complex was shown to betranscriptionally active and contained both ATP-dependent 5′-3′(contributed by XPD) and 3′-5′ (contributed by XPB) helicase activitiesby using the helicase assays described above. This system of assaysallows us to simultaneously assay and distinguish between XPD and XPBhelicase activity within the native BTF2-TFIIH complex. (see FIG. 5).

[0083] A highly purified human wild-type recombinant p53 proteinproduced from baculovirus effectively was shown to inhibit intrinsicBTF2-TFIIH helicase activity in a dose-dependent manner (FIG. 5A, bothorientations, compare lanes 5-7 to lanes 3 and 4; FIG. 5C). XPD wasinhibited to a greater degree than XPB (FIG. 5C). No inhibition wasobserved with the addition of mutant p53-273H (FIG. 5A, comparing lanes9-10 to lanes 3 and 4; FIG. 5C), even though it binds to XPD and XPBproteins (see FIG. 1). Thus, wild-type p53 was demonstrated to inhibithelicase activity XPD and XPB, while mutant p53 did not inhibit thisactivity.

[0084] It has been reported that wild-type p53 binds preferentially tothe ends of single-stranded (SS) DNA or RNA in vitro, and catalyzes DNAor RNA strand reassociation. While not wishing to be bound by theory,inhibition of the helicase activity of BTF2-TFIIH and Rad3 in our systemdoes not appear to be due to the strand annealing activity of p53, butrather due to direct interaction with these factors. This conclusion isbased on the following data. First, p53 selectively binds to XPD andXPB, which are the major components of the 5′→13′ and 3′→5′ DNA helicaseactivities of BTF2-TFIIH. Second, p53 at a similar concentration used inthe BTF2-TFIIH helicase assay inhibits Rad3 helicase activity to asimilar extent with a substrate containing a circular ss DNA that doesnot preferentially bind to p53. Third, inhibition of the BTF2-TFIIHhelicase activity is not uniform for XPD and XPB, i.e., XPD is affectedto a greater degree than XPB (see FIG. 5C).

Example 7

[0085] Induction of Apoptosis in Normal Human Fibroblasts by p53

[0086] In order to demonstrate that increased levels of wt p53 aresufficient to induce apoptosis in normal human fibroblasts, we used amicroinjection technique to deliver an expression vector encoding wt p53under the control of the cytomegalovirus early promoter (CMV) into thenuclei of homopolykaryons of fused normal human primary fibroblasts(C5RO). These homopolykaryons have lost proliferation potential and arearrested primarily in G1 of the cell cycle (see Vermeulen, W., et al.(1994) Am. J. Hum. Genet. 54:191). Cell strains, culture conditions,cell hybridization and microinjection were as described in Vermeulen,W., et al. (1994) Am. J. Hum. Genet. 54:191. Primary human fibroblastswere grown in Ham's F10 medium supplemented with 10% FBS and fused withβ-propiolactone-inactivated Sendai virus. Fused cells were seeded ontocoverslip and incubated for additional 2-3 days prior to injection.Plasmid cDNA in a concentration of 100-200 μg/ml suspended in PBS wasinjected into one of the nuclei of homopolykaryons by using a glassmicrocapillary. For each experiment, at least 50 homopolykaryons wereinjected. Following incubation, cells were fixed with 2%paraformaldehyde (in PBS) followed by methanol treatment. p53 wasvisualized by staining cells with CM-1 antibody (Signet Labs, Dedham,Mass., U.S.A.) followed by fluorescein conjugated anti-rabbit IgG(Vector Labs, Burlingame, Calif., USA). Nuclei were stained with4′,6-diamidino-2-phenylindole (DAPI). The results are shown in FIG. 7and summarized in Table 1.

[0087] High levels of wt p53 were observed 24 hrs following injection,predominantly in the nucleus or in both the nucleus and cytoplasm (FIG.1A, Table 1) and 20% of the cells displayed the typical characteristicfeatures of apoptosis, including chromatin condensation, nuclearfragmentation and apoptotic bodies (FIG. 1B and data not shown). Thispercentage is an underestimate since apoptotic bodies staining positivefor p53 were visible when most cells and debris were detached from theplates (data not shown), and these residual cells were not scored. TABLE1 Induction of apoptosis by wild-type or mutant p53 in normal primaryhuman fibroblasts Expression % apoptotic p53 localization (% cells)Vectors cells^(a) (n)^(b) nuclei cytoplasmic both WT p53 20 (152) 28 072 143^(ala) 0(18) 11 6 83 175^(his) 0(38) 26 24 50 248^(trp) 0(36) 44 848 249^(ser) 0(34) 6 77 17 273^(his) 7(30) 27 0 73 WT + 143^(ala) 0(16)13 6 81 WT + 175^(his) 0(17) 12 47 41 WT + 248^(trp) 0(24) 63 16 21 WT +249^(ser) 0(20) 45 20 35 WT + 273^(his) 0(15) 33 33 34

[0088] To demonstrate that induction of apoptosis was due specificallyto an intrinsic activity of wt p53 and not the result of nonspecificprotein overproduction, p53 mutants, 143^(ala), 175^(his), 248^(trp),249^(ser) and 273^(his), were microinjected in the same expressionsystem. These p53 mutants all lack the sequence-specific transcriptionalactivation activity that is associated with growth suppression. Allmutants were expressed at similar or higher levels of protein whencompared to the wt p53 (FIGS. 1C, 1E and unpublished data). The p53mutants showed similar subcellular localization with both nuclear andcytoplasmic staining, except for 249^(ser), that accumulatedpredominantly in the cytoplasm (FIGS. 1C, 1E, Table 1). The 273^(his)mutant behaved similar to wt p53 in cellular localization (Table 1). Incontrast to wt p53, mutants 143^(ala), 175^(his), 248^(trp) and249^(ser) also exhibited only cytoplasmic localization in some cellsranging from 6 to 77% (Table 1), indicating that these mutants may havea tendency to accumulate in cytoplasm. All mutants exhibited eithersignificantly reduced (273]^(his)) or no ability to induce apoptosis(Table 1).

[0089] The data summarized in Table 1 indicate that an intrinsicproperty of wt p53 is the induction of apoptosis. Mutant 273^(his) stillretained wt-like activity to a small degree (subcellular localizationand ability to induce apoptosis), suggesting that it is a weak mutant.While the 273^(his) mutant has none of transactivating function that isimportant in inducing G1 growth arrest, it could still induce apoptosis,suggesting that these two events can be uncoupled. Supporting theseobservations are the findings that mutant 273^(his) retains wtconformation as measured by PAb1620 recognition, lacks Hsp70 binding,and exhibits partial tumor suppressor activity. Other recent studiesalso indicate that cell cycle arrest and apoptosis may be independentoutcomes following treatment with cytotoxic agents.

[0090] Mutations of the wt p53 gene result not only in loss of tumorsuppressor activity but also gain of oncogenic activity. In order todemonstrate dominant negative effects of mutant p53, expression vectorsencoding both wt and mutant p53 were coinjected into normal humanfibroblasts as described above. The presence of the mutants, i.e.,143^(ala), 175^(his), 248^(trp), 249^(ser) or 273^(his), completelyabolished the induction of apoptosis by p53 (Table 1). The subcellularlocalization patterns of most mutants were unaltered when co-expressedwith wt p53. Interestingly, we observed a lesser number of cells withonly cytoplasmic staining when wt p53 was co-injected with 249^(ser)(20% vs 77%), but increased percentage of cells with only cytoplasmicstaining when wt p53 was co-injected with 273^(his) (33% vs 0%). Thesedata indicate that although all p53 mutants tested exhibit dominantnegative effects, they may differ in their pathways to inactivate the wtp53 function. While not wishing to be bound by theory, these results areconsistent with the hypothesis that regulation of apoptosis by wt p53 isan integral part of the defense mechanism against outgrowth of damagedcells which may lead to cancer development.

Example 8

[0091] Lack of Inhibition of Apoptosis With wt p53

[0092] As described herein, both wt p53 and mutant p53 (135^(tyr),249^(ser) and 273^(his)) bind to TFIIH-associated factors, but only wtp53 inhibits the TFIIH-based DNA helicase activity contributed by XPBand XPD. Furthermore, as we demonstrate below, defects in the XPD andXPB genes particularly associated with the helicase activity result incells resistant to p53-induced apoptosis. The cells from patient XPCS2BAcontain a missense mutation at codon 99^(phe→ser) in the XPB gene,resulting in virtually complete inactivation of the NER function of theprotein. (See Vermeulen, W., et al. (1994) Am. J. Hum. Genet. 54:191.The cells from patient XP6BE contain a defective XPD gene that alsoabolishes NER activity. (See Flejter, W. L., et al. (1992) Proc. Natl.Acad. Sci U.S.A. 89:261.) Microinjection of the wt p53 expression vectorinto XPCS2BA or XP6BE cells and measurement of apoptosis was carried outas described in Example 7. Results are shown in table 2. TABLE 2Differential induction of apoptosis between normal primary humanfibroblasts and fibroblasts from XPB and XPD patients Cell % apoptoticp53 localization (% cells) strains cells^(a) (n)^(b) nuclei cytoplasmicboth Normal 20 (152) 28 0 72 XPCS2BA  4(113) 22 2 76 (XPB) XP6BE (XPD)0(95) 40 0 60

[0093] Microinjection of the wt p53 expression vector into XPCS2BA orXP6BE cells resulted in expression of high levels of nuclear p53 (Table2). The signal intensity of p53 in these cells is comparable to thelevels in normal fibroblasts (C5RO) (data not shown). Although 20% ofC5RO cells underwent apoptosis, only 4% of the p53-positive XPCS2BAcells and none of the XP6BE cells exhibited apoptosis at 24 hr (Table2).

[0094] We then carried out a time-course study comparing different timepoints after microinjection of p53 into XPCS2BA or XP6BE cells. Cellswere incubated for 6, 24, and 48 hr following microinjection of wt p53expression vector. High levels of p53 could be detected at 6 hrfollowing injection in all cell types although no apoptosis wasobserved. At 24 hr, 20% of C5RO cells, but only 4% of XPCS2BA cells hadundergone apoptosis (Table 2, FIG. 2). No apoptosis was observed inXP6BE cells, as described above. At 48 hr, however, 33% of both C5RO andXPCS2BA cells and 9% of XP6BE cells had undergone apoptosis (FIG. 2). Itappears that the p53 associated apoptosis is not completely abolishedalthough a difference in timing, and possibly efficiency of apoptosiscan be seen. While not wishing to be bound by theory, these differencesmay be due to the XPB and XPD dependent apoptosis pathways beingdeficient, but still marginally functioning resulted from the complex ofthese proteins. These differences may also reflect other signallingpathways which follow a different time course to trigger apoptosis.

[0095] In order to further demonstrate that p53 induced apoptosis infibroblasts is mediated by effects on XPB and XPD, fibroblasts fromindividuals with defects in repair genes other than XPB and XPD werealso examined. In particular, we infected fibroblasts from normalindividuals and Xeroderma pigmentosum donors with XP-A and XP-C germlinemutation by wt p53 in a retroviral expression vector under the controlof CMV promoter. Infection of the p53 retroviral expression vectorresulted in a moderate levels of nuclear p53 proteins expressed in allcell types tested as compared to microinjection (data not shown). Theeffect on apoptosis is shown in Table 3. TABLE 3 Differential inductionof apoptosis in fibroblasts from individuals with various defects innucleotide excision repair pathway by infection of a retroviral vectorencoding wt p53 % apoptosis (n^(b)) Cell Name^(a) Phenotypes Exp I ExpII GM07532/1057 Normal  6.4 (204) 11.0 (91)  GM00510/XP1PW XPC 7.1 (98)7.4 (244) GM05509B/XP12BE XPA 4.5 (67) 5.2 (155) GM13025/XPCS2BA XPB 1.3(78) 0.9 (217) GM10430/XP6BE XPD   0 (51)   0 (168) # Research betweenpassage 10 and 15. The cell names correspond to their # catalog numbersor their local user numbers that were used in the initial #publications. XP-A, XP-B, XP-D, and XP-C stand for Xeroderma #pigmentosum complementation group A, B, D, and C, respectively.Induction # of apoptosis was achieved by infection of these cells with aretroviral # vector encoding wt p53 under the control of CMV promoter,followed by an # additional 48 hr incubation. The titer of viral stocksused is between # 5 × 10⁴ to 1 × 10⁵ cfu/ml. Cells were fixed, stainedfor p53, and # analyzed for apoptosis as described in Example 8, herein.Two independent # experiments were performed on a separate day, and werereferred to as Exp I or Exp II.

[0096] About 9% of GM7532 cells (normal), 5% of GM5509 cells (XPA), and7% of GM0510 cells (XPC) consistently exhibited apoptosis (Table 3). Incontrast, only 1% of XPCS2BA cells (XPB) and none of XP6BE cells (XPD)showed apoptosis (Table 3), which is consistent with the microinjectiondata. These results indicate that decreased sensitivity to wtp53-induced apoptosis in XP-B and XP-D cells is not due to a generaloverall defect in NER pathway but rather as a result of a mutation inthe XPB or XPD gene.

[0097] Our results show for the first time that the association of p53and the TFIIH (XPB/XPD) complex has functional consequence in vivo, andprovide a basis for defining down-stream targets in p53 dependentapoptosis. It is already known that XPB and XPD are part of the TFIIHcomplex that are necessary for both basal transcription and NER. Thefunctions of these proteins may depend on their physical associationsince they always form a complex in vivo. While not wishing to be boundby theory, it is possible that these three pathways have a number ofsteps in common using the same proteins. Furthermore, it is likely thatp53 induces apoptosis by binding to and inhibiting the helicaseactivities of both the XPD and XPB gene products. Therefore, XP-D orXP-B cells that contain a defect only in XPD or XPB gene, respectively,are not completely resistant to the p53-induced apoptosis.

[0098] The findings that p53 inhibits DNA helicases associated with NERas well as DNA and RNA helicases involved in DNA replication andtranslation suggest that p53 may influence a broad range of cellularprocesses. Although the unwinding reaction of helicases is poorlyunderstood, these enzymes are in fact essential for all aspects of DNAfunction, including DNA replication, repair, recombination,transcription, and translation. As described herein, p53 binds to XPB ata region within helicase motif III that may be essential for nucleicacid unwinding and is indispensable for its NER activity. Sequence andcomputer-aided secondary structure analysis of motif III from manymembers of the helicase superfamily indicate that they are structurallyand functionally related, and could be the targets of p53.Correspondingly, p53 may act as a modulator of cellular helicaseactivity. It is generally believed, however, that the loss of p53 leadsto genomic instability along with failure to undergo DNA damage-inducedapoptosis, a defense mechanism preventing cells from surviving DNAdamage that may result in the acquisition of mutations conferringuncontrolled growth advantage. Thus, while not wishing to be bound bytheory, the general anti-helicase activity of p53 could be part of thesignal transduction pathway associated with apoptosis upon DNA damage.

[0099] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

1 6 393 amino acids amino acid single linear protein Protein 1..393/note= “human wild-type p53” 1 Met Glu Glu Pro Gln Ser Asp Pro Ser ValGlu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys LeuLeu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro Ser Gln Ala Met AspAsp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu AspPro Gly Pro Asp Glu Ala Pro 50 55 60 Arg Met Pro Glu Ala Ala Pro Arg ValAla Pro Gly Pro Ala Ala Pro 65 70 75 80 Thr Pro Ala Ala Pro Ala Pro AlaPro Ser Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr GlnGly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu His Ser Gly Thr AlaLys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met PheCys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135 140 Leu Trp Val Asp SerThr Pro Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile TyrLys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys 165 170 175 Pro HisHis Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190 HisLeu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195 200 205Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu 210 215220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser 225230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile IleThr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser PheGlu Val 260 265 270 Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr GluGlu Glu Asn 275 280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu ProPro Gly Ser Thr 290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser SerPro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe ThrLeu Gln Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu Met Phe Arg Glu LeuAsn Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu ProGly Gly Ser Arg Ala His Ser Ser His 355 360 365 Leu Lys Ser Lys Lys GlyGln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu GlyPro Asp Ser Asp 385 390 21 amino acids amino acid <Unknown> linearpeptide Peptide 1..21 /note= “peptide # p53cp from amino acid residues367-387 of human wild-type p53 capable of inhibiting binding ofwild-type p53 to XPB” 2 Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr SerArg His Lys Lys 1 5 10 15 Leu Met Phe Lys Thr 20 781 amino acids aminoacid <Unknown> linear protein Protein 1..781 /note= “human xerodermapigmentosum B (XPB) helicase protein” 3 Met Gly Lys Arg Asp Arg Ala AspArg Asp Lys Lys Lys Ser Arg Lys 1 5 10 15 Arg His Tyr Glu Asp Glu GluAsp Asp Glu Glu Asp Ala Pro Gly Asn 20 25 30 Asp Pro Gln Glu Ala Val ProSer Ala Ala Gly Lys Gln Val Asp Glu 35 40 45 Ser Gly Thr Lys Val Asp GluTyr Gly Ala Lys Asp Tyr Arg Leu Gln 50 55 60 Met Pro Leu Lys Asp Asp HisThr Ser Arg Pro Leu Trp Val Ala Pro 65 70 75 80 Asp Gly His Ile Phe LeuGlu Ala Phe Ser Pro Val Tyr Lys Tyr Ala 85 90 95 Gln Asp Phe Leu Val AlaIle Ala Glu Pro Val Cys Arg Pro Thr His 100 105 110 Val His Glu Tyr LysLeu Thr Ala Tyr Ser Leu Tyr Ala Ala Val Ser 115 120 125 Val Gly Leu GlnThr Ser Asp Ile Thr Glu Tyr Leu Arg Lys Leu Ser 130 135 140 Lys Thr GlyVal Pro Asp Gly Ile Met Gln Phe Ile Lys Leu Cys Thr 145 150 155 160 ValSer Tyr Gly Lys Val Lys Leu Val Leu Lys His Asn Arg Tyr Phe 165 170 175Val Glu Ser Cys His Pro Asp Val Ile Gln His Leu Leu Gln Asp Pro 180 185190 Val Ile Arg Glu Cys Arg Leu Arg Asn Ser Glu Gly Glu Ala Thr Glu 195200 205 Leu Ile Thr Glu Thr Phe Thr Ser Lys Ser Ala Ile Ser Lys Thr Ala210 215 220 Glu Ser Ser Gly Gly Pro Ser Thr Ser Arg Val Thr Asp Pro GlnGly 225 230 235 240 Lys Ser Asp Ile Pro Met Asp Leu Phe Asp Phe Tyr GluGln Met Asp 245 250 255 Lys Asp Glu Glu Glu Glu Glu Glu Thr Gln Thr ValSer Phe Glu Val 260 265 270 Lys Gln Glu Met Ile Glu Glu Leu Gln Lys ArgCys Ile His Leu Glu 275 280 285 Tyr Pro Leu Leu Ala Glu Tyr Asp Phe ArgAsn Asp Ser Val Asn Pro 290 295 300 Asp Ile Asn Ile Asp Leu Lys Pro ThrAla Val Leu Arg Pro Tyr Gln 305 310 315 320 Glu Lys Ser Leu Arg Lys MetPhe Gly Asn Gly Arg Ala Arg Ser Gly 325 330 335 Val Ile Val Leu Pro CysGly Ala Gly Lys Ser Leu Val Gly Val Thr 340 345 350 Ala Ala Cys Thr ValArg Lys Arg Cys Leu Val Leu Gly Asn Ser Ala 355 360 365 Val Ser Val GluGln Trp Lys Ala Gln Phe Lys Met Trp Ser Thr Ile 370 375 380 Asp Asp SerGln Ile Cys Arg Phe Thr Ser Asp Ala Lys Asp Lys Pro 385 390 395 400 IleGly Cys Ser Val Ala Ile Ser Thr Tyr Ser Met Leu Gly His Thr 405 410 415Thr Lys Arg Ser Trp Glu Ala Glu Arg Val Met Glu Trp Leu Lys Thr 420 425430 Gln Glu Trp Gly Leu Met Ile Leu Asp Glu Val His Thr Ile Pro Ala 435440 445 Lys Met Phe Arg Arg Val Leu Thr Ile Val Gln Ala His Cys Lys Leu450 455 460 Gly Leu Thr Ala Thr Leu Val Arg Glu Asp Asp Lys Ile Val AspLeu 465 470 475 480 Asn Phe Leu Ile Gly Pro Lys Leu Tyr Glu Ala Asn TrpMet Glu Leu 485 490 495 Gln Asn Asn Gly Tyr Ile Ala Lys Val Gln Cys AlaGlu Val Trp Cys 500 505 510 Pro Met Ser Pro Glu Phe Tyr Arg Glu Tyr ValAla Ile Lys Thr Lys 515 520 525 Lys Arg Ile Leu Leu Tyr Thr Met Asn ProAsn Lys Phe Arg Ala Cys 530 535 540 Gln Phe Leu Ile Lys Phe His Glu ArgArg Asn Asp Lys Ile Ile Val 545 550 555 560 Phe Ala Asp Asn Val Phe AlaLeu Lys Glu Tyr Ala Ile Arg Leu Asn 565 570 575 Lys Pro Tyr Ile Tyr GlyPro Thr Ser Gln Gly Glu Arg Met Gln Ile 580 585 590 Leu Gln Asn Phe LysHis Asn Pro Lys Ile Asn Thr Ile Phe Ile Ser 595 600 605 Lys Val Gly AspThr Ser Phe Asp Leu Pro Glu Ala Asn Val Leu Ile 610 615 620 Gln Ile SerSer His Gly Gly Ser Arg Arg Gln Glu Ala Gln Arg Leu 625 630 635 640 GlyArg Val Leu Arg Ala Lys Lys Gly Met Val Ala Glu Glu Tyr Asn 645 650 655Ala Phe Phe Tyr Ser Leu Val Ser Gln Asp Thr Gln Glu Met Ala Tyr 660 665670 Ser Thr Lys Arg Gln Arg Phe Leu Val Gln Gly Tyr Ser Phe Lys Val 675680 685 Ile Thr Lys Leu Ala Gly Met Glu Glu Glu Asp Leu Ala Phe Ser Thr690 695 700 Lys Glu Glu Gln Gln Gln Leu Leu Gln Lys Val Leu Ala Ala ThrAsp 705 710 715 720 Leu Asp Ala Glu Glu Glu Val Val Ala Gly Glu Phe GlySer Arg Ser 725 730 735 Ser Gln Ala Ser Arg Arg Phe Gly Thr Met Ser SerMet Ser Gly Ala 740 745 750 Asp Asp Thr Val Tyr Met Glu Tyr His Ser SerArg Ser Lys Ala Pro 755 760 765 Ser Lys His Val His Pro Leu Phe Lys ArgPhe Arg Lys 770 775 780 15 amino acids amino acid <Unknown> linearpeptide Peptide 1..15 /note= “peptide # 464 from amino acid residues464-478 of the helicase III region of XPB protein capable of inhibitingbinding of wild-type p53 to XPB” 4 Leu Gly Leu Thr Ala Thr Leu Val ArgGlu Asp Asp Lys Ile Val 1 5 10 15 15 amino acids amino acid <Unknown>linear peptide Peptide 1..15 /note= “peptide # 479 from amino acidresidues 479-493 of XPB protein incapable of inhibiting binding ofwild-type p53 to XPB” 5 Asp Leu Asn Phe Leu Ile Gly Pro Lys Leu Tyr GluAla Asn Trp 1 5 10 15 16 amino acids amino acid <Unknown> linear peptidePeptide 1..16 /note= “peptide # 99 irrelevant peptide from HBV” 6 GlyLeu Ser Ala Met Ser Thr Thr Asp Leu Glu Ala Tyr Phe Lys Asp 1 5 10 15

What is claimed is:
 1. A method for screening a compound for an abilityto induce apoptosis comprising: a) providing a first cell containing anormal or mutant p53 gene, wherein said first cell is capable ofundergoing apoptosis after microinjection of a DNA construct expressingwild type p53; b) providing a second cell containing at least one of amutant XPB gene and a mutant XPD gene, wherein said second cell is lesscapable than said first cell of undergoing apoptosis aftermicroinjection of a DNA construct expressing wild type 53; c) contactingeach of the first cell and the second cell with the compound; d)detecting whether or not apoptosis of the first cell occurs; e)detecting whether or not apoptosis of the second cell occurs; and f)comparing the detectings of steps (d) and (e), thereby determiningwhether the compound can induce apoptosis.
 2. A method of claim 1further comprising the step of selecting at least one of the first celland the second cell from the group consisting of fibroblastic,epithelial, and hematopoietic cells.
 3. A method of screening for acompound capable of inhibiting the binding of p53 protein to at leastone of XPB and XPD proteins comprising: (a) providing a reagent havingat least one of XPB and XPD; (b) contacting the reagent with thecompound, permitting the compound to compete with wild type p53 proteinfor a binding site on at least one of XPB and XPD proteins; and (c)detecting a binding of the compound to at least one of XPB and XPDproteins.
 4. A method of claim 3 further comprising contacting thereagent with wild type p53 protein and detecting a binding of the wildtype p53 to at least one of XPB and XPD proteins.
 5. A method of claim 3further comprising attaching a label to at least one of the XPB, XPD,and p53 proteins.
 6. A method of claim 5 wherein the label is selectedfrom the group consisting an antibody, a radioisotope, and a fluorescentmolecule.
 7. A method of claim 3 wherein the reagent has a TFIIH complexcontaining both XPB and XPD proteins.
 8. A method of screening for acompound capable of inhibiting at least one of XPB and XPD helicaseactivity comprising: (a) providing a reagent having at least one of XPBand XPD proteins; (b) contacting the reagent with the compound,permitting the compound to bind to at least one of XPB and XPD helicase;and (c) determining the helicase activity.
 9. A method of claim 8wherein the reagent has a TFIIH complex containing both XPB and XPDproteins.
 10. A compound consisting essentially of the amino acidsequence depicted in Seq. ID No. 2, wherein said compound (1) binds to abinding site on at least one of the XPB helicase and the XPD helicase,(2) competes with wild type p53 proteins for the binding site, and (3)inhibits the helicase activity.
 11. A compound of claim 10 wherein thecompound is a peptide consisting of the sequence depicted in Seq. ID No.2.
 12. A method of diagnosing Xeroderma pigmentosum complementationgroup B or D in an individual comprising: (a) providing a sample cellderived from the individual; b) contacting the sample cell with thecompound of claim 10; and c) detecting whether or not apoptosis of thesample cell occurs, thereby diagnosing whether or not the sample cellcontains at least one of a mutant XPB gene and a mutant XPD gene.
 13. Acompound consisting essentially of the amino acid sequence depicted inSeq. ID No. 4 wherein said compound (1) binds to a binding site on wildtype p53 protein and (2) competitively inhibits the binding of wild typep53 protein to wild type XPB protein.
 14. A compound of claim 13 whereinthe compound consists of the amino acid sequence depicted in Seq ID No.4.
 15. A method of diagnosing Xeroderma pigmentosum complementationgroup B or D in an individual comprising: (a) providing a sample cellderived from the individual; b) contacting the sample cell with thecompound of claim 13; and c) detecting whether or not apoptosis of thesample cell occurs, thereby diagnosing whether or not the sample cellcontains at least one of a mutant XPB gene and a mutant XPD gene.